The present invention relates to the methods and nucleic acid compositions for the production of transgenic plants resistant to virus infection. In particular, it relates to transgenic plants comprising nucleotide sequences encoding dysfunctional viral movement protein (dMP) genes.
Local as well as systemic viral infection requires virus movement. Opportunistic introduction of viral particles occurs where cell wall and plasma membrane integrity has been disrupted, as for example through mechanical damage caused by a biological vector such as an insect, nematode or fungus as-well-as abrasive forces such as the breaking of a leaf or branch. A progressive viral infection results if upon replication the viral progeny is capable of spreading into adjacent cells and then systemically throughout the plant. In certain instances infectious virus have been shown as capable of replication in the host cells but unable to move to adjacent healthy cells. When this occurs the infection is said to be subliminal and the plant appears resistant.
The cell-to-cell spread of virus is not a passive process but requires the expression of a virus encoded product called a movement protein (MP). The movement proteins of many viruses have been tentatively identified as reviewed by Hull, R., Annu Rev Phytopathol 27: 213-240 1989, and Maule, A. J., Crit Tev Plant Sci 9: 457-473 1991. The first virus-encoded movement protein (MP) identified was that of tobacco mosaic virus (TMV) (Deom et al., Science, 237 384-389 1987; and Meshi et al., EMBO J 6: 2557-2563 1987). Although dispensable for virus replication, the 30 kDa MP of TMV is essential for cell-to-cell spread of the infection (Deom et al., Cell 69: 221-224 1992). Furthermore, transgenic plants that express the TMV MP (MP(+) plants) can complement mutants of TMV that are movement deficient (Deom et al., supra 1987; Holt and Beachy, Virology 181: 109-117 1991). In both TMV-infected plants and MP(+) plants the MP co-purifies with an insoluble cellular component that contains cell walls (Deom et al., Proc Natl Acad Sci 87: 3284-3288, 1990), and was localized by immunogold labeling to the plasmodesmata, the cytoplasmic connections between adjacent plant cells. Specifically, the TMV MP is localized to the central cavity of secondary (also referred to as modified primary) plasmodesmata which are formed through fusion of, and addition of, new protoplasmic bridges to primary plasmodesmata (Ding et al., Plant Cell, 4: 915-928, 1992).
The mechanism(s) by which the MP potentiates virus movement from cell to cell is not fully understood. Wolf et al., Science 246: 377-379, 1989, demonstrated through dye coupling studies that the protein has a direct effect on the molecular size exclusion limit (SEL) of the plasmodesmata. Fluorescent dextrans with an average molecular mass of 9400 Da moved between cells of MP(+) transgenic plants, while the size exclusion limit of the MP(-) transgenic plants and non-transgenic plants was 700-800 Da. In a similar study a temperature-sensitive (ts) mutant of the MP was unable to modify the SEL of plasmodesmata or to facilitate virus movement at the non-permissive temperature (Wolf et al., Plant Cell 3: 593-604, 1991).
Deletion mutants of TMV MP have been made and expressed in transgenic plants. Berna et al., Virology, 182: 682-689, 1991, studied transgenic plants that expressed truncated MP lacking up to 73 C-terminal amino acids. The transformed plants were analyzed for MP subcellular localization and for complementation of the spread of a thermosensitive TMV mutant Ls1. Ls1 is incapable of cell-to-cell movement at the non-permissive (32.degree. C.) temperature due to an inactivated MP. Deletion of the C-terminal 55 amino acids of MP had no effect on subcellular localization or complementation of Ls1. Deletion of an additional 19 aa (aa 195 to 213 aa) destroyed both cell localization and ability to complement Ls1.
Gafny, et al., Virology 187: 499-507 1992, studied a variety of infectious clones of TMV having N-terminal and C-terminal deletions in the MP gene. The effect of the deletion mutations on local and systemic movements of the infection was evaluated. Deletion of 9 to 33 C-terminal amino acids did not effect cell-to-cell movement as reflected by local lesion formation on Nicotiana tabacum cv. Xanthi NN plants. Deletion of 55 C-terminal amino acids resulted in impaired movement and deletion of 74 C-terminal amino acids resulted in a protein that could not support virus movement. In addition, MP deleted for N-terminal amino acids 3-5 could not support virus movement.
Although a number of mutations in the TMV MP are found to alter the ability of the protein to support virus spread, the effect such mutations have on the production of a MP capable of blocking the spread of virus infection was not determined. It is the determination that dysfunctional MP can lead to viral resistance in plants, the resultant dysfunctional MP and the virus resistant transgenic plants that are the subject of the present invention.